During the previous funding period we showed that glioma cells shrink as they invade the narrow extracellular spaces in the brain. Cell shrinkage is energetically driven by K+ and Cl- efflux through ion channels, which osmotically drives water out of the cell. We identified the underlying Cl-, and Ca2+-activated K+ channels and demonstrated that their pharmacological inhibition renders glioma cells unable to invade. One of the underlying channels, ClC-3, is regulated by chlorotoxin, a scorpion-derived peptide that inhibits Cl- currents and retards glioma invasion. These finding led us to initiate a Phase I/II clinical trial examining chlorotoxin as an anti-tumor drug in patients with malignant glioma which is now in Phase II. This competitive renewal application expands on unexpected observations made that mechanistically link Cl- movement to cell volume changes that occur in the context of cell proliferation and terminal cell proliferation, i.e. apoptosis. Specifically we hypothesize that glioma cells maintain elevated intracellular Cl- through active ion transport, which in turn allows cells to release Cl- through channels as they condense to enter mitosis. Cell shrinkage is directly caused by Cl- efflux and its inhibition inhibits proliferation. It also prevents condensation preceding apoptosis. Following each cell division cells re- establish their original volume through transport mediated Cl-/K+ and water uptake, which again is a necessary step for cell growth. 4 Specific aims are proposed that study the underlying Cl- channels and transporters, their mechanistic role in cell volume changes associated with proliferation and apoptosis as well as their regulation by phosphorylation and membrane trafficking. Finally, two FDA approved Cl- transport inhibitors will be examined preclinically using an animal model for malignant glioma. PUBLIC HEALTH RELEVANCE: This competitive renewal application expands on unexpected observations made that mechanistically link Cl- movement to cell volume changes that occur in the context of cell proliferation and terminal cell proliferation, i.e. apoptosis.